Gene therapy consists in correcting a deficiency or an abnormality by introducing genetic information into the affected cell or organ. This information may be introduced either in vitro into a cell extracted from the organ and then reinjected into the body, or in vivo, directly into the tissue concerned. Being a high molecular weight, negatively charged molecule, DNA has difficulties in passing spontaneously through the phospholipid cell membranes. Different vectors are hence used in order to permit gene transfer: viral vectors on the one hand, natural or synthetic, chemical and/or biochemical vectors on the other hand. Viral vectors (retroviruses, adenoviruses, adeno-associated viruses, etc.) are very effective, in particular in passing through membranes, but present a number of risks, such as pathogenicity, recombination, replication, immunogenicity, etc. Chemical and/or biochemical vectors enable these risks to be avoided (for reviews, see Behr, 1993, Cotten and Wagner, 1993). These vectors are, for example, cations (calcium phosphate, DEAE-dextran, etc.) which act by forming precipitates with DNA, which precipitates can be xe2x80x9cphagocytosedxe2x80x9d by the cells. They can also be liposomes in which DNA is incorporated and which fuse with the plasma membrane. Synthetic gene transfer vectors are generally lipids or cationic polymers which complex DNA and form a particle therewith carrying positive surface charges. These particles are capable of interacting with the negative charges of the cell membrane and then of crossing the latter. Dioctadecylamidoglycylspermine (DOGS, Transfectam(trademark)) or N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA, Lipofectin(trademark)) may be mentioned as examples of such vectors. Chimeric proteins have also been developed: they consist of a polycationic portion which condenses DNA, linked to a ligand which binds to a membrane receptor and carries the complex into the cells by endocytosis. It is thus theoretically possible to xe2x80x9ctargetxe2x80x9d a tissue or certain cell populations so as to improve the in vivo bioavailability of the transferred gene.
However, the use of chemical and/or biochemical vectors or of naked DNA implies the possibility of producing large amounts of DNA of pharmacological purity. In effect, in these gene therapy techniques, the medicinal product consists of the DNA itself, and it is essential to be able to manufacture, in appropriate amounts, DNAs having suitable properties for therapeutic use in man.
The plasmids currently used in gene therapy carry (i) an origin of replication, (ii) a marker gene such as a gene for resistance to an antibiotic (kanamycin, ampicillin, etc.) and (iii) one or more transgenes with sequences required for their expression (enhancer(s), promoter(s), polyadenylation sequences, etc.). These plasmids currently used in gene therapy (in clinical trials such as the treatment of melanomas, Nabel et al., 1992, or in experimental studies) display, however, some drawbacks associated, in particular, with their dissemination in the body. Thus, as a result of this dissemination, a competent bacterium present in the body can, at a low frequency, receive this plasmid. The chance of this occurring is all the greater for the fact that the treatment in question entails in vivo gene therapy in which the DNA may be disseminated in the patient""s body and may come into contact with bacteria which infect this patient or alternatively with bacteria of the commensal flora. If the bacterium which is a recipient of the plasmid is an enterobacterium such as E. coli, this plasmid may replicate. Such an event then leads to the dissemination of the therapeutic gene. Inasmuch as the therapeutic genes used in gene therapy treatments can code, for example, for a lymphokine, a growth factor, an anti-oncogene, or a protein whose function is lacking in the host and hence enables a genetic defect to be corrected, the dissemination of some of these genes could have unforeseeable and worrying effects (for example if a pathogenic bacterium were to acquire the gene for a human growth factor). Furthermore, the plasmids used in non-viral gene therapy also possess a marker for resistance to an antibiotic (ampicillin, kanamycin, etc.). Hence the bacterium acquiring such a plasmid has an undeniable selective advantage, since any therapeutic antibiotic treatment using an antibiotic of the same family as the one selecting the resistance gene of the plasmid will lead to the selection of the plasmid in question. In this connection, ampicillin belongs to the xcex2-lactams, which is the family of antibiotics most widely used in the world. It is hence necessary to seek to limit as far as possible the dissemination of the therapeutic genes and the resistance genes. Moreover, the genes carried by the plasmid, corresponding to the vector portion of the plasmid (function(s) required for replication, resistance gene), also run the risk of being expressed in the transfected cells. There is, in effect, a transcription background, which cannot be ruled out, due to the host""s expression signals on the plasmid. This expression of exogenous proteins may be thoroughly detrimental in a number of gene therapy treatments, as a result of their potential immunogenicity and hence of the attack of the transfected cells by the immune system.
Hence it is especially important to be able to have at one""s disposal medicinal DNA molecules having a genetic purity suitable for therapeutic use. It is also especially important to have at one""s disposal methods enabling these DNA molecules to be prepared in amounts appropriate for pharmaceutical use. The present invention provides a solution to these problems.
The present invention describes, in effect, DNA molecules which can be used in gene therapy, having greatly improved genetic purity and impressive properties of bioavailability. The invention also describes an especially effective method for the preparation of these molecules and for their purification.
The present invention lies, in particular, in the development of DNA molecules which can be used in gene therapy, virtually lacking any non-therapeutic region. The DNA molecules according to the invention, also designated minicircles on account of their circular structure, their small size and their-supercoiled form, display many advantages.
They make it possible, in the first place, to eliminate the risks associated with dissemination of the plasmid, such as (1) replication and dissemination which may lead to an uncontrolled overexpression of the therapeutic gene, (2) the dissemination and expression of resistance genes, and (3) the expression of genes present in the non-therapeutic portion of the plasmid, which are potentially immunogenic and/or inflammatory, and the like. The genetic information contained in the DNA molecules according to the invention is limited, in effect, essentially to the therapeutic gene(s) and to the signals for regulation of its/their expression (neither origin of replication, nor gene for resistance to an antibiotic, and the like). The probability of these molecules (and hence of the genetic information they contain) being transferred to a microorganism and being stably maintained is almost zero.
Furthermore, due to their small size, DNA molecules according to the invention potentially have better bioavailability in vivo. In particular, they display improved capacities for cell penetration and cellular distribution. Thus, it is recognized that the coefficient of diffusion in the tissues is inversely proportional to the molecular weight (Jain, 1987). Similarly, at cellular level, high molecular weight molecules have inferior permeability through the plasma membrane. In addition, for the plasmid to progress to the nucleus, which is essential for its expression, high molecular weight is also a drawback, the nuclear pores imposing a size limit for diffusion to the nucleus (Landford et al., 1986). The elimination of the non-therapeutic portions of the plasmid (origin of replication and resistance gene in particular) according to the invention also enables the size of the DNA molecules to be decreased. This decrease may be estimated at a factor of 2, reckoning, for example, 3 kb for the origin of replication and the resistance marker (vector portion) and 3 kb for the transgene with the sequences required for its expression. This decrease (i) in molecular weight and (ii) in negative charge endows the molecules of the invention with improved capacities for tissue, cellular and nuclear diffusion and bioavailability.
Hence a first subject of the invention lies in a double-stranded DNA molecule having the following features: it is circular in shape and essentially comprises one or more genes of interest. As stated above, the molecules of the invention essentially lack non-therapeutic regions, and especially an origin of replication and/or a marker gene. In addition, they are advantageously in supercoiled form.
The present invention is also the outcome for the development of a method, of constructions and of cell hosts which are specific and especially effective for the production of these therapeutic DNA molecules. More especially, the method according to the invention lies in the production of therapeutic DNA molecules defined above, by excision from a plasmid or from a chromosome by site-specific recombination. The method according to the invention is especially advantageous, since it does not necessitate a prior step of purification of the plasmid, is very specific, especially effective, does not decrease the amounts of DNA produced and leads directly to therapeutic molecules of very great genetic purity and of great bioavailability. This method leads, in effect, to the generation of circular DNA molecules (minicircles) essentially containing the gene of interest and the regulator sequences permitting its expression in the cells, tissue, organ or apparatus, or even the whole body, in which the expression is desired. In addition, these molecules may then be purified by standard techniques.
The site-specific recombination may be carried out by means of various systems which lead to site-specific recombination between sequences. More preferably, the site-specific recombination in the method of the invention is obtained by means of two specific sequences which are capable of recombining with one another in the presence of a specific protein, generally designated recombinase. For this reason, the DNA molecules according to the invention generally comprise, in addition, a sequence resulting from this site-specific recombination. The sequences permitting the recombination used in the context of the invention generally comprise from 5 to 100 base pairs, and more preferably fewer than 50 base pairs.
The site-specific recombination may be carried out in vivo (that is to say in the host cell) or in vitro (that is to say on a plasmid preparation).
In this connection, the present invention also provides particular genetic constructions suitable for the production of the therapeutic DNA molecules defined above. These genetic constructions, or recombinant DNAs, according to the invention comprise, in particular, the gene or genes of interest flanked by the two sequences permitting site-specific recombination, positioned in the direct orientation. The position in the direct orientation indicates that the two sequences follow the same 5xe2x80x2-3xe2x80x2 polarity in the recombinant DNA according to the invention. The genetic constructions of the invention can be double-stranded DNA fragments (cassettes) essentially composed of the elements mentioned above. These cassettes can be used for the construction of cell hosts having these elements integrated in their genome (FIG. 1). The genetic constructions of the invention can also be plasmids, that is to say any linear or circular DNA molecule capable of replicating in a given host cell, containing the gene or genes of interest flanked by the two sequences permitting site-specific recombination, positioned in the direct orientation. The construction can be, more specifically, a vector (such as a cloning and/or expression vector), a phage, a virus, and the like. These plasmids of the invention may be used to transform any competent cell host for the purpose of the production of minicircles by replication of the plasmid followed by excision of the minicircle (FIG. 2).
In this connection, another subject of the invention lies in a recombinant DNA comprising one or more genes of interest, flanked by two sequences permitting site-specific recombination, positioned in the direct orientation.
The recombinant DNA according to the invention is preferably a plasmid comprising at least:
a) an origin of replication and optionally a marker gene,
b) two sequences permitting a site-specific recombination, positioned in the direct orientation, and,
c) placed between said sequences b), one or more genes of interest.
The specific recombination system present in the genetic constructions according to the invention can be of different origins, in particular, the specific sequences and the recombinases used can belong to different structural classes, and in particular to the integrase family of bacteriophage xcex or to the resolvase family of the transposon Tn3.
Among recombinases belonging to the integrase family of bacteriophage xcex, there may be mentioned, in particular, the integrase of the phages lambda (Landy et al., Science 197 (1977) 1147), P22 and "PHgr"80 (Leong et al., J. Biol. Chem. 260 (1985) 4468), HP1 of Haemophilus influenza (Hauser et al., J. Biol. Chem. 267 (1992) 6859), the Cre integrase of phage P1, the integrase of the plasmid pSAM2 (EP 350,341) or alternatively the FLP recombinase of the 2xcexc plasmid. When the DNA molecules according to the invention are prepared by recombination by means of a site-specific system of the integrase family of bacteriophage lambda, the DNA molecules according to the invention generally comprise, in addition, a sequence resulting from the recombination between two att attachment sequences of the corresponding bacteriophage or plasmid.
Among recombinases belonging to the family of the transposon Tn3, there may be mentioned, in particular, the resolvase of the transposon Tn3 or of the transposons Tn21 and Tn522 (Stark et al., 1992); the Gin invertase of bacteriophage mu or alternatively the resolvase of plasmids, such as that of the par fragment of RP4 (Albert et al., Mol. Microbiol. 12 (1994) 131). When the DNA molecules according to the invention are prepared by recombination by means of a site-specific system of the family of the transposon Tn3, the DNA molecules according to the invention generally comprise, in addition, a sequence resulting from the recombination between two recognition sequences of the resolvase of the transposon in question.
According to a particular embodiment, in the genetic constructions of the present invention, the sequences permitting site-specific recombination are derived from a bacteriophage. More preferably, these latter are attachment sequences (attP and attB sequences) of a bacteriophage, or derived sequences. These sequences are capable of recombining specifically with one another in the presence of a recombinase designated integrase. The term derived sequence includes the sequences obtained by modification(s) of the attachment sequences of the bacteriophages, which retain the capacity to recombine specifically in the presence of the appropriate recombinase. Thus, such sequences can be reduced fragments of these sequences or, on the contrary, fragments extended by the addition of other sequences (restriction sites, and the like). They can also be variants obtained by mutation(s), in particular by point mutation(s). The terms attP and attB sequences of a bacteriophage or of a plasmid denote, according to the invention, the sequences of the recombination system specific to said bacteriophage or plasmid, that is to say the attP sequence present in said phage or plasmid and the corresponding chromosomal attB sequence.
By way of preferred examples, there may be mentioned, in particular, the attachment sequences of the phages lambda, P22, "PHgr"80, P1 and HP1 of Haemophilus influenzae or alternatively of plasmid pSAM2 or the 2xcexc plasmid. These sequences are advantageously chosen from all or part of the sequences SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13 and SEQ ID No. 14. These sequences comprise, in particular, the central region homologous to the attachment sequences of these phages.
In this connection, a preferred plasmid according to the present invention comprises
(a) a bacterial origin of replication and optionally a marker gene,
(b) the attP and attB sequences of a bacteriophage selected from the phages lambda, P22, "PHgr"80, HP1 and P1 or of plasmid pSAM2 or the 2xcexc plasmid, or derived sequences; and,
(c) placed between said sequences b), one or more genes of interest.
According to an especially preferred embodiment, the sequences in question are the attachment sequences (attP and attB) of phage lambda. Plasmids carrying these sequences are, in particular, the plasmids pXL2648, pXL2649 or pXL2650. When these plasmids are brought, in vivo or in vitro, into contact with the integrase of phage lambda, the sequences recombine with one another to generate in vivo or in vitro, by excision, a minicircle according to the invention essentially comprising the elements (c), that is to say the therapeutic portion (FIG. 2).
Still according to a particular embodiment of the invention, the sequences permitting site-specific recombination are derived from the loxP region of phage P1. This region is composed essentially of two inverted repeat sequences capable of recombining specifically with one another in the presence of a protein, designated Cre (Sternberg et al., J. Mol. Biol. 150 (1971) 467). In a particular variant, the invention hence relates to a plasmid comprising (a) a bacterial origin of replication and optionally a marker gene; (b) the inverted repeat sequences of bacteriophage P1 (loxP region); and (c) placed between said sequences (b), one or more genes of interest.
According to another particular embodiment, in the genetic constructions of the present invention, the sequences permitting site-specific recombination are derived from a transposon. More preferably, the sequences in question are recognition sequences of the resolvase of a transposon, or derived sequences. By way of preferred examples, there may be mentioned, in particular, the recognition sequences of the transposons Tn3, Tn21 and Tn522. By way of a preferred example, there may be mentioned the sequence SEQ ID No. 15 or a derivative of the latter (see also Sherrat, P. 163-184, Mobile DNA, Ed. D. Berg and M. Howe, American Society for Microbiology, Washington D.C. 1989).
According to another especially advantageous variant, the plasmids of the invention comprise, in addition, a multimer resolution sequence. This is preferably the mrs (multimer resolution system) sequence of the plasmid RK2. More preferably, the invention relates to a plasmid comprising:
(a) a bacterial origin of replication and optionally a marker gene,
(b) the attP and attB sequences of a bacteriophage, in the direct orientation, selected from the phages lambda, P22, "PHgr"80, HP1 and P1 or of plasmid pSAM2 or the 2xcexc plasmid, or derived sequences; and,
(c) placed between said sequences b), one or more genes of interest and the mrs sequence of plasmid RK2.
This embodiment is especially advantageous. Thus, when plasmids pXL2649 or pXL2650 are brought into contact with the integrase of the bacteriophage in vivo, the sequences recombine to generate the minicircle and the miniplasmid, but also multimeric or topological forms or minicircle or of miniplasmid. It is especially advantageous to be able to decrease the concentration of these forms in order to increase the production and facilitate the purification of minicircle.
The multimeric forms of plasmids are known to a person skilled in the art. For example, the cer fragment of ColE1 (Summers et al., 1984 Cell 36 p. 1097) or the mrs site of the par locus of RK2 (L. Ebert 1994 Mol. Microbiol. 2 p. 131) permit the resolution of multimers of plasmids and participate in an enhanced stability of the plasmid. However, whereas resolution at the cer site requires four proteins encoded by the E. coli genome (Colloms et al., 1990 J. Bacteriol. 172 p. 6973), resolution at the mrs site requires only the ParA protein for which the parA gene is mapped on the Dar locus of RK2. As a result, it would appear advantageous to use all or a portion of the par locus containing parA and the mrs sequence. For example, the mrs sequence may be placed between the attB and attP sequences of phage lambda, and the parA gene be expressed in trans or in cis from its own promoter or from an inducible promoter. In this connection, a particular plasmid of the invention comprises:
(a) a bacterial origin of replication and optionally a marker gene,
(b) the attP and attB sequences of a bacteriophage, in the direct orientation, selected from the phages lambda, P22, "PHgr"80, HP1 and P1 or of plasmid pSAM2 or the 2xcexc plasmid, or derived sequences,
(c) placed between said sequences b), one or more genes of interest and the mrs sequence of plasmid RK2, and
(d) the parA gene of plasmid RK2.
One such plasmid is, in particular, the plasmid pXL2960 described in the examples. It may be employed, and can enable minicircle to be produced exclusively in monomeric form.
According to another advantageous variant, the plasmids of the invention comprise two sets of site-specific recombination sequences from a different family. These advantageously comprise a first set of integrase-dependent sequences and a second set of parA-dependent sequences. The use of two sets of sequences enables the production yields of minicircles to be increased when the first site-specific recombination is incomplete. Thus, when plasmids pXL2650 or pXL2960 are brought into contact with the integrase of the bacteriophage in vivo, the sequences recombine to generate the miniplasmid and the minicircle, but this reaction is not complete (5 to 10% of initial plasmid may be left). The introduction, in proximity to each of the att sequences of phage lambda, of an mrs sequence of RK2 enables the production of minicircles to be increased. Thus, after induction of the integrase of phage lambda and Int-dependent recombination, the unrecombined molecules will be able to come under the control of the ParA protein of RK2 and to recombine at the mrs sites. Conversely, after induction of the ParA protein and ParA-dependent recombination, the unrecombined molecules will be able to come under the control of the integrase of phage lambda and will be able to recombine at the att sites. Such constructions thus make it possible to produce minicircle and negligible amounts of unrecombined molecules. The att sequences, like the mrs sequences, are in the direct orientation, and the int and parA genes may be induced simultaneously or successively from the same inducible promoter or from two inducible promoters. Preferably, the sequences in question are the attB and attP attachment sequences of phage lambda in the direct orientation and two mrs sequences of RK2 in the direct orientation.
As stated above, another aspect of the present invention lies in a method for the production of therapeutic DNA molecules defined above, by excision, from a plasmid or chromosome, by site-specific recombination.
Another subject of the present invention hence lies in a method for the production of a DNA molecule (minicircle) as defined above, according to which a culture of host cells containing a recombinant DNA as defined above is brought into contact with the recombinase enabling site-specific recombination to be induced. More preferably, the culture and recombinase are brought into contact either by transfection or infection with a plasmid or a phage containing the gene for said recombinase; or by induction of the expression of a gene coding for said recombinase, present in the host cell. As mentioned below, this gene may be present in the host cell in integrated form in the genome, on a replicative plasmid or alternatively on the plasmid of the invention, in the non-therapeutic portion.
To permit the production of the minicircles according to the invention by site-specific recombination in vivo, the recombinase used must be introduced into, or induced in, cells or the culture medium at a particular instant. For this purpose, different methods may be used. According to a first method, a host cell is used containing the recombinase gene in a form permitting its regulated expression. It may, in particular, be introduced under the control of a promoter or of a system of inducible promoters, or alternatively in a temperature-sensitive system. In particular, the gene may be present in a temperature-sensitive phage, latent during the growth phase, and induced at a suitable temperature (for example lysogenic phage lambda Xisxe2x88x92 cI857). The cassette for expression of the recombinase gene may be carried by a plasmid, a phage or even by the plasmid of the invention, in the non-therapeutic region. It may be integrated in the genome of the host cell or maintained in replicative form. According to another method, the cassette for expression of the gene is carried by a plasmid or a phage used to transfect or infect the cell culture after the growth phase. In this case, it is not necessary for the gene to be in a form permitting its regulated expression. In particular, any constitutive promoter may be used. The cell may also be brought into contact with the recombinase in vitro, on a plasmid preparation, by direct incubation with the protein.
It is preferable, in the context of the present invention, to use a host cell capable of expressing the recombinase gene in a regulated manner. This embodiment, in which the recombinase is supplied directly by the host cell after induction, is especially advantageous. In effect, it suffices simply to place the cells in culture at the desired time under the conditions for expression of the recombinase gene (permissive temperature for a temperature-sensitive gene, addition of an inducer for a regulable promoter, and the like) in order to induce the site-specific recombination in vivo and thus the excision of the minicircle of the invention. In addition, this excision takes place in especially high yields, since all the cells in culture express the recombinase, which is not necessarily the case if a transfection or an infection has to be carried out in order to transfer the recombinase gene.
According to a first embodiment, the method of the invention comprises the excision of the molecules of therapeutic DNA by site-specific recombination from a plasmid. This embodiment employs the plasmids described above permitting, in a first stage, replication in a chosen host, and then, in a second stage, the excision of the non-therapeutic portions of said plasmid (in particular the origin of replication and the resistance gene) by site-specific recombination, generating the circular DNA molecules of the invention. To carry out the method, different types of plasmid may be used, and especially a vector, a phage or a virus. A replicative vector is preferably used.
Advantageously, the method of the invention comprises a prior step of transformation of host cells with a plasmid as defined above, followed by culturing of the transformed cells, enabling suitable amounts of plasmid to be obtained. Excision by site-specific recombinations is then carried out by bringing into contact with the recombinase under the conditions defined above (FIG. 2). As stated above, in this embodiment, the site-specific recombination may be carried out in vivo. (that is to say in the host cell) or in vitro (that is to say on a plasmid preparation).
According to a preferred embodiment, the DNA molecules of the invention are hence obtained from a replicative vector, by excision of the non-therapeutic portion carrying, in particular, the origin of replication and the marker gene, by site-specific recombination.
According to another embodiment, the method of the invention comprises the excision of the DNA molecules from the genome of the host cell by site-specific recombination. This embodiment is based more especially on the construction of cell hosts comprising, inserted into their genome, one or more copies of a cassette comprising the gene of interest flanked by the sequences permitting recombination (FIG. 1). Different techniques may be used for insertion of the cassette of the invention into the genome of the host cell. In particular, insertion at several distinct points of the genome may be obtained by using integrative vectors. In this connection, different transposition systems such as, in particular, the miniMu system or defective transposons such as Tn10 derivatives, for example, may be used (Kleckner et al., Methods Enzymol. 204 (1991) 139; Groisman E., Methods Enzymol. 204 (1991) 180). The insertion may also be carried out by homologous recombination, enabling a cassette containing two recombination sequences in the direct orientation flanking one or more genes of interest to be integrated in the genome of the bacterium. This process may, in addition, be reproduced as many times as desired so as to have the largest possible number of copies per cell. Another technique also consists in using an in vivo amplification system using recombination, as described in Labarre et al. (Labarre J., 0. Reyes, Guyonvarch, and G. Leblon. 1993. Gene replacement, integration, and amplification at the gdhA locus of Corynebacterium glutamicum. J. Bacteriol. 175:1001-107), so as to augment from one copy of the cassette to a much larger number.
A preferred technique consists in the use of miniMu. To this end, miniMu derivatives are constructed comprising a resistance marker, the functions required in cis for their transposition and a cassette containing two recombination sequences in the direct orientation flanking the gene or genes of interest. These miniMus are advantageously placed at several points of the genome using a resistance marker (kanamycin, for example) enabling several copies per genome to be selected (Groisman E. cited above). As described above, the host cell in question can also express inducibly a site-specific recombinase leading to the excision of the fragment flanked by the recombination sequences in the direct orientation. After excision, the minicircles may be purified by standard techniques.
This embodiment of the method of the invention is especially advantageous, since it leads to the generation of a single type of plasmid molecule: the minicircle of the invention. The cells do not contain, in effect, any other episomal plasmid, as is the case during production from a plasmid (FIGS. 1 and 2).
Another subject of the invention also lies in a modified host cell comprising, inserted into its genome, one or more copies of a recombinant DNA as defined above. The invention also relates to any recombinant cell containing a plasmid as defined above. These cells are obtained by any technique known to a person skilled in the art enabling a DNA to be introduced into a given cell. Such a technique can be, in particular, transformation, electroporation, conjugation, protoplast fusion or any other technique known to a person skilled in the art.
As regards transformation, different protocols have been described in the prior art. In particular, cell transformation may be carried out by treating whole cells in the presence of lithium acetate and polyethylene glycol according to the technique described by Ito et al. (J. Bacteriol. 153 (1983) 163-168), or in the presence of ethylene glycol and dimethyl sulphoxide according to the technique of Durrens et al. (Curr. Genet. 18 (1990) 7). An alternative protocol has also been described in Patent Application EP 361,991. As regards electroporation, this may be carried out according to Becker and Guarentte (in: Methods in Enzymology Vol194 (1991) 182).
The method according to the invention may be carried out in any type of cell host. Such hosts can be, in particular, bacteria or eukaryotic cells (yeasts, animal cells, plant cells), and the like. Among bacteria, E. coli, B. subtilis, Streptomyces, Pseudomonas (P. putida, P. aeruginosa), Rhizobium meliloti, Agrobacterium tumefaciens, Staphylococcus aureus, Streptomyces pristinesoiralis, Enterococcus faecium or Clostridium, and the like, may be mentioned more preferentially. Among bacteria, it is preferable to use E. coli. Among yeasts, Kluyveromyces, Saccharomyces, Pichia, Hansenula, and the like, may be mentioned. Among mammalian animal cells, CHO, COS, NIH3T3, and the like, cells may be mentioned.
In accordance with the host used, the plasmid according to the invention is adapted by a person skilled in the art to permit its replication. In particular, the origin of replication and the marker gene are chosen in accordance with the host cell selected.
The marker gene may be a resistance gene, in particular for resistance to an antibiotic (ampicillin, kanamycin, geneticin, hygromycin, and the like), or any gene endowing the cell with a function which it no longer possesses (for example a gene which has been deleted on the chromosome or rendered inactive), the gene on the plasmid reestablishing this function.
In a particular embodiment, the method of the invention comprises an additional step of purification of the minicircle.
In this connection, the minicircle may be purified by standard techniques of plasmid DNA purification, since it is supercoiled like plasmid DNA. These techniques comprise, inter alia, purification on a caesium chloride density gradient in the presence of ethidium bromide, or alternatively the use of anion exchange columns (Maniatis et al., 1989). In addition, if the plasmid DNA corresponding to the non-therapeutic portions (origin of replication and selectable marker in particular) is considered to be present in an excessively large amount, it is also possible, after or before the purification, to use one or more restriction enzymes which will digest the plasmid and not the minicircle, enabling them to be separated by techniques that separate supercoiled DNA from linear DNA, such as a caesium chloride density gradient in the presence of ethidium bromide (Maniatis et al., 1989).
In addition, the present invention also describes an improved method for the purification of minicircles. This method enables minicircles of very great purity to be obtained in large yields in a single step. This improved method is based on the interaction between a double-stranded sequence present in the minicircle and a specific ligand. The ligand can be of various natures, and in particular protein, chemical or nucleic acid in nature. It is preferably a ligand of the nucleic acid type, and in particular an oligonucleotide, optionally chemically modified, capable of forming by hybridization a triple helix with the specific sequence present in the DNA molecule of the invention. It was, in effect, shown that some oligonucleotides were capable of specifically forming triple helices with double-stranded DNA sequences (Hxc3xa9lxc3xa8ne et al., Biochim. Biophys. Acta 1049 (1990) 99; see also FR 94/15162 incorporated in the present application by reference).
In an especially advantageous variant, the DNA molecules of the invention hence contain, in addition, a sequence capable of interacting specifically with a ligand (FIG. 3). Preferably, it is a sequence capable of forming, by hybridization, a triple helix with a specific oligonucleotide. This sequence may be positioned at any site of the DNA molecule of the invention, provided it does not affect the functionality of the gene of interest. This sequence is also present in the genetic constructions of the invention (plasmids, cassettes), in the portion containing the gene of interest (see, in particular, the plasmid pXL2650). Preferably, the specific sequence present in the DNA molecule of the invention comprises between 5 and 30 base pairs.
The oligonucleotides used for carrying out the method according to the invention can contain the following bases:
thymidine (T), which is capable of forming triplets with A.T doublets of double-stranded DNA Rajagopal et al., Biochem 28 (1989) 7859);
adenine (A), which is capable of forming triplets with A.T doublets of double-stranded DNA;
guanine (G), which is capable of forming triplets with G.C doublets of doubled-stranded DNA;
protonated cytosine (C+), which is capable of forming triplets with G.C, doublets of doubled-stranded DNA (Rajagopal et al., cited above).
Preferably, the oligonucleotide used comprises a homopyrimidine sequence containing cytosines, and the specific sequence present in the DNA molecule is a homopurine-homopyrimidine sequence. The presence of cytosines makes it possible to have a triple helix which is stable at acid pH where the cytosines are protonated, and destabilized at alkaline pH where the cytosines are neutralized.
To permit the formation of a triple helix by hybridization, it is important for the oligonucleotide and the specific sequence present in the DNA molecule of the invention to be complementary. In this connection, to obtain the best yields and best selectivity, an oligonucleotide and a specific sequence which are fully complementary are used in the method of the invention. Possible combinations are, in particular, a poly(CTT) oligonucleotide and a poly(GAA) specific sequence. By way of example, there may be mentioned the oligonucleotide of sequence GAGGCTTCTTCTTCTTCTTCTTCTT (SEQ ID No. 5), in which the bases GAGG do not form a triple helix but enable the oligonucleotide to be spaced apart from the coupling arm.
It is understood, however, that some mismatches may be tolerated, provided they do not lead to too great a loss of affinity. The oligonucleotide used may be natural (composed of unmodified natural bases) or chemically modified. In particular, the oligonucleotide may advantageously possess some chemical modifications enabling its resistance or its protection against nucleases, or its affinity for the specific sequence, to be increased.
Thus, the oligonucleotide may be rendered more resistant to nucleases by modification of the skeleton (e.g. methylphosphonates, phosphorothiates, phosphotriester, phosphoramidate, and the like). Another type of modification has as its objective, more especially, to improve the interaction and/or the affinity between the oligonucleotide and the specific sequence. In particular, a thoroughly advantageous Modification according to the invention consists in methylating the cytosines of the oligonucleotide. The oligonucleotide thus methylated displays the noteworthy property of forming a stable triple helix with the specific sequence at neutral pH.
Hence it makes it possible to work at higher pH values than the oligonucleotides of the prior art, that is to say at pH values where the risks of degradation of the plasmid DNA are lower.
The length of the oligonucleotide used in the method of the invention is at least 3 bases, and preferably between 5 and 30. An oligonucleotide of length greater than 10 bases is advantageously used. The length may be adapted to each individual case by a person skilled in the art in accordance with the desired selectivity and stability of the interaction.
The oligonucleotides according to the invention may be synthesized by any known technique. In particular, they may be prepared by means of nucleic acid synthesizers. It is quite obvious that any other method known to a person skilled in the art may be used.
To carry out the method of the invention, the specific ligand (protein, nucleic acid, and the like) may be grafted or otherwise onto a support. Different types of supports may be used for this purpose, such as, in particular, functionalized chromatography supports, in bulk form or prepacked in columns, functionalized plastic surfaces or functionalized latex beads, magnetic or otherwise. Chromatography supports are preferably used. By way of example, the chromatography supports which may be used are agarose, acrylamide or dextran, as well as their derivatives (such as Sephadex, Sepharose, Superose, etc.), polymers such as poly(styrenedivinylbenzene), or grafted or ungrafted silica, for example. The chromatography columns can function in the diffusion or perfusion mode.
To permit its covalent coupling to the support, the ligand is generally functionalized. In the case of an oligonucleotide, this may be modified, for example, with a terminal thiol, amine or carboxyl group at the 5xe2x80x2 or 3xe2x80x2 position. In particular, the addition of a thiol, amine or carboxyl group makes it possible, for example, to couple the oligonucleotide to a support carrying disulphide, maleimide, amine, carboxyl, ester, epoxide, cyanogen bromide or aldehyde functions. These couplings form by the establishment of disulphide, thioether, ester, amide or amine links between the oligonucleotide and the support. Any other method known to a person skilled in the art may be used, such as bifunctional coupling reagents, for example.
Moreover, to improve the activity of the coupled oligonucleotide, it may be advantageous to perform the coupling by means of an xe2x80x9carmxe2x80x9d. Use of an arm makes it possible, in effect, to bind the oligonucleotide at a chosen distance from the support, enabling its conditions of interaction with the DNA molecule of the invention to be improved. The arm advantageously consists of nucleotide bases that do not interfere with the hybridization. Thus, the arm may comprise purine bases. By way of example, the arm may comprise the sequence GAGG.
The DNA molecules according to the invention may be used in any application of vaccination or of gene and cell therapy, for the transfer of a gene to a body, a tissue or a given cell. In particular, they may be used for a direct administration in vivo, or for the modification of cells in vitro or ex vivo with a view to their implantation in a patient. In this connection, the molecules according to the invention may be used as they are (in the form of naked DNA), or in combination with different synthetic or natural, chemical and/or biochemical vectors. The latter can be, in particular, cations (calcium phosphate, DEAE-dextran, etc.) which act by forming precipitates with DNA, which precipitates can be xe2x80x9cphagocytosedxe2x80x9d by the cells. They can also be liposomes in which the DNA molecule is incorporated and which fuse with the plasma membrane. Synthetic gene transfer vectors are generally lipids or cationic polymers which complex DNA and form a particle, therewith carrying positive surface charges. These particles are capable of interacting with the negative charges of the cell membrane and then of crossing the latter. DOGS (Transfectam(trademark)) or DOTMA (Lipofectin(trademark)) may be mentioned as examples of such vectors. Chimeric proteins have also been developed: they consist of a polycationic portion which condenses DNA, linked to a ligand which binds to a membrane receptor and carries the complex into the cells by endocytosis. The DNA molecules according to the invention may also be used for gene transfer into cells by physical transfection techniques such as bombardment, electroporation, and the like. In addition, prior to their therapeutic use, the molecules of the invention may optionally be linearized, for example by enzymatic cleavage.
In this connection, another subject of the present invention relates to any pharmaceutical composition comprising at least one DNA molecule as defined above. This molecule may be naked or combined with a chemical and/or biochemical transfection vector. The pharmaceutical compositions according to the invention may be formulated with a view to topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intra-ocular, transdermal, and the like, administration. Preferably, the DNA molecule is used in an injectable form or by application. It may be mixed with any pharmaceutically acceptable vehicle for an injectable formulation, in particular for a direct injection at the site to be treated. The compositions can be, in particular, in the form of isotonic sterile solutions, or of dry, in particular lyophilized compositions which, on addition of sterilized water or physiological saline as appropriate, enable injectable solutions to be made up. Diluted Tris or PBS buffers in glucose or sodium chloride may be used in particular. A direct injection of the nucleic acid into the affected region of the patient is advantageous, since it enables the therapeutic effect to be concentrated in the tissues affected. The doses of nucleic acid used may be adapted in accordance with different parameters, and in particular in accordance with the gene, the vector, the mode of administration used, the pathology in question or alternatively the desired treatment period.
The DNA molecules of the invention may contain one or more genes of interest, that is to say one or more nucleic acids (cDNA, gDNA, synthetic or semi-synthetic DNA, and the like) whose transcription and, where appropriate, translation in the target cell generate products of therapeutic, vaccinal, agricultural or veterinary value.
Among the genes of therapeutic value, there may be mentioned, more especially, the genes coding for enzymes, blood derivatives, hormones, lymphokines, namely interleukins, interferons, TNF, and the like (FR 92/03120), growth factors, neurotransmitters or their precursors or synthetic enzymes, trophic factors, namely BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, and the like; apolipoproteins, namely ApoAI, ApoAIV, ApoE, and the like (FR 93/05125), dystrophin or a minidystrophin (FR 91/11947), tumour suppressive genes, namely p53, Rb, Rap1A, DCC, k-rev, and the like (FR 93/04745), genes coding for factors involved in coagulation, namely factors VII, VIII, IX, and the like, suicide genes, namely thymidine kinase, cytosine deaminase, and the like; or alternatively all or part of a natural or artificial immunoglobulin (Fab, ScFv, and the like), a ligand RNA (WO91/19813), and the like. The therapeutic gene can also be an antisense gene or sequence whose expression in the target cell enables gene expression or the transcription of cellular mRNAs to be controlled. Such sequences can, for example, be transcribed in the target cell into RNAs complementary to cellular mRNAs, and can thus block their translation into protein, according to the technique described in Patent EP 140,308.
The gene of interest can also be a vaccinating gene, that is to say a gene coding for an antigenic peptide, capable of generating an immune response in man or animals for the purpose of vaccine production. Such antigenic peptides can be, in particular, those specific to the Epstein-Barr virus, the HIV virus, the hepatitis B virus (EP 185,573) or the pseudorabies virus, or alternatively tumour-specific peptides (EP 259,212).
Generally, in the plasmids and molecules of the invention, the gene of therapeutic, vaccinal, agricultural or veterinary value also contains a transcription promoter region which is functional in the target cell or body (i.e. mammals), as well as a region located at the 3xe2x80x2 end and which specifies a transcription termination signal and a polyadenylation site (expression cassette). As regards the promoter region, this can be a promoter region naturally responsible for the expression of the gene in question when the latter is capable of functioning in the cell or body in question. The promoter regions can also be those of different origin (responsible for the expression of other proteins, or even synthetic promoters). In particular, the promoter sequences can be from eukaryotic or viral genes. For example, they can be promoter sequences originating from the genome of the target cell. Among eukaryotic promoters, it is possible to use any promoter or derived sequence that stimulates or represses the transcription of a gene, specifically or otherwise, inducibly or otherwise, strongly or weakly. They can be, in particular, ubiquitous promoters (promoter of the HPRT, PGK, xcex1-actin, tubulin, and the like, genes), promoters of intermediate filaments (promoter of the GFAP, desmin, vimentin, neurofilament, keratin, and the like, genes), promoters of therapeutic genes (for example the promoter of the MDR, CFTR, factor VIII, ApoAI, and the like, genes), tissue-specific promoters (promoter of the pyruvate kinase gene, villin gene, gene for intestinal fatty acid binding protein, gene for xcex1-actin of smooth muscle, and the like) or alternatively promoters that respond to a stimulus (steroid hormone receptor, retinoic acid receptor, and the like). Similarly, the promoter sequences may be those originating from the genome of a virus, such as, for example, the promoters of the adenovirus E1A and MLP genes, the CMV early promoter or alternatively the RSV LTR promoter, and the like. In addition, these promoter regions may be modified by the addition of activator or regulator sequences or sequences permitting a tissue-specific or -preponderant expression.
Moreover, the gene of interest can also contain a signal sequence directing the synthesized product into the pathways of secretion of the target cell. This signal sequence can be the natural signal sequence of the product synthesized, but it can also be any other functional signal sequence, or an artificial signal sequence.
Depending on the gene of interest, the DNA molecules of the invention may be used for the treatment or prevention of a large number of pathologies, including genetic disorders (dystrophy, cystic fibrosis, and the like), neurodegenerative diseases (Alzheimer""s, Parkinson""s, ALS, and the like), cancers, pathologies associated with disorders of coagulation or with dyslipoproteinaemias, pathologies associated with viral infections (hepatitis, AIDS, and the like), or in the agricultural and veterinary fields, and the like.
The present invention will be described more completely by means of the examples which follow, which are to be regarded as illustrative and non-limiting.